Project Summary/Abstract The overall goal of this project is to understand the cell biophysical mechanisms regulating transmembrane signal transduction, i.e. signal transfer from ligand to receptor to downstream effector. There is mounting evidence that cell surface receptors exhibit a high degree of dynamic organization, yet little is known about the mechanisms underlying this organization and its consequences for receptor signaling. This project will focus on vascular endothelial growth factor receptor 2 (VEGFR2), a key receptor in endothelial cells promoting angiogenesis, the process of sprouting new blood vessels from the existing vasculature. Angiogenesis is critical for growth and development, and goes astray in many diseases, from cancer to ischemia. In addition to the pathophysiological importance of angiogenesis, there are many commonalities between VEGFR2 signaling and other signaling pathways. Thus the knowledge gained from the proposed studies is expected to be applicable beyond the specifics of angiogenesis. This project will focus on the following major questions: (1) What mechanisms regulate the cell surface spatiotemporal organization and signaling of VEGFR2? The goal here is to determine the membrane and cytosolic factors that regulate VEGFR2 spatiotemporal organization on the cell surface (i.e. its dynamics, oligomerization state and spatial distribution), and to test the hypothesis that these factors provide a mechanism for the cell to modulate VEGFR2?s response to VEGF. (2) How do inter-receptor interactions regulate VEGFR2 signaling in response to its ligand VEGF? The goal here is to quantitatively characterize VEGFR2 interactions with other receptors, starting with the antagonistic anti-angiogenic receptor CD36, and to test the hypothesis that these interactions contribute to the integration of pro- and anti-angiogenic signals. (3) What are the spatiotemporal characteristics of signal transfer from VEGFR2 to downstream effectors? The goal here is to determine the nanoscale spatial relationship and kinetics of signal transfer from VEGFR2 to its downstream effectors, starting with phosphoinositide-3-kinase (PI3K). This will allow us to quantitatively link the spatiotemporal organization of VEGFR2 to its functional consequences. These questions will be addressed by developing integrative approaches combining cellular light microscopy (single-molecule, super-resolution and activity biosensor imaging) with novel analytical tools (computational image analysis, statistical data analysis and mathematical modeling). These analytical tools are necessary to extract quantitative, complete information from each imaging modality and to rigorously multiplex the complementary information that the different modalities reveal. Together they will enable the monitoring of VEGFR2 spatiotemporal organization and interactions down to the single-molecule level, and quantitatively link it to VEGFR2 signal initiation in its native cellular context. These integrative approaches and tools will be also broadly applicable, contributing to the biomedical research community beyond this specific project.